The invention discloses a nanopore-based in vitro HIV protease detector and method. The detector comprises a blood sample/body fluid separation chip (1), a micro-fluidic chip (2), a nanopore detectioncircuit system (3) and a system collection control system (4), wherein a blood sample/body fluid injection port (101) receives a blood sample/body fluid sample, theblood sample/body fluid sample flows into a mixing channel (103) to mix with a pyrolysis solution flowing into a pyrolysis channel (102), a mixed solution passes a filter membrane (104), then large molecular samples are filtered and flow into a waste liquid pool (106), HIV protease (205) small molecules that are not filtered penetrate the filter membrane and flow into a separated sample outflow port (105) so as to be mixed and react with pre-added target protein (202), two short fragments (206 and 207) generate via signals, the content of the HIV protease is calculated, and finally, the content is displayed in computer or a cellphone.